PST-16

Original link: https://science.mahidol.ac.th/pst2021/viweposter.php

17-Nov-2021

Engineered decoy receptor

He wants to block the interaction between spike protein of virus + host dimeric ACE2
His lab perform thousand of mutagenesis of ACE2 decoy -- to inhibit the interaction
He expressed the decoy with different mutations on the human cell lines
Screening for the one having the high affinity to viral spike protein
Picking up the one containing high affinity and run molecular dynamics simulation to explain the interaction between the engineered decoy and viral protein
One thing that they learn is that the sequence which responsible for host ACE2 binding is diverse but the structural conformation is the same
He also performed the viability assay with the mouse expressing hACE2

Developing the inhibitor toward SARS-CoV-2 main protease

He collaborated with computational chemistry in order to understand the interaction between protein and drug
He wants to get the new inhibitor as the alternative route to against viral protease, not only the wild-type but also the
He heterologous expressed the main protease from E. coli and trying to setting up the bioassay + to do crystallography
For the bioassay, using the peptide tagged with fluorescence -- using FRET technique to assay the cleavage of the peptide
Using computational chemistry to get the inhibitors from natural compounds (Thai herbs) to inhibit the protease
He used ANS as a probe to determine the inhibition activity rather than using the peptide-tag substrate -- ANS will fluoresce when binding to hydrophobic pocket (in this case, the binding pocket of protease is hydrophobic -- thus he used this approach to detect the competitive inhibitor) -- this approach is for compensating the short supply of peptide substrate due to COVID-19 situation
Lower in fluorescence means that the inhibitor competes with ANS.
Flavonoid is the real pain in the neck -- it causes PAN-assay interference (I used to hear this word from the reviewer of Biomed and Pharm J.)

Engineered survival-selective strategy for synthetic binding proteins

20-25,000 genes -- > ~100,000 transcripts -- > post-translational modification ~ a million unique molecules.
FLI-TRAP

Using E. coli cytosol as the platform to screen proper folded protein.
Making use of Tat machinery in E. coli to export the correct folded protein to periplasm. If the protein is misfolded, it could not be exported through this pathway.
He genetic engineered both binder and receptor and set up the reaction in the cytosol of bacteria, however, there are some limitations in term of selecting the binder -- for example, the binder should not contain disulfide bond
The target is tagged with beta-lactamase which if being exported together with its interaction partner (binders) -- E. coli develops resistance toward ampicillin.
The binder is tagged with Tat-signal in order to force the whole complex to be exported through the tat-related transporter system -- under the condition that the whole complex must fold properly.

New inhibitor targeting sugar binding enzyme

Lectins -- glycocode -- reading the glycan-conjugated
From the structure, no glycan bs with glycan active pocket -- > the same. No glycan crystal structure, more water molecules occupy in the pocket. When superimposed with structure with glycan in the pocket, the water molecules are at the same position where the sugar forming the bonds with the pocket site.
Focusing on Pseudomonas aeruginosa lectins -- this bacteria is in the priority list of antibiotic development according to WHO report.
This bacteria also developed the current use of antibiotics. Thus, there is an urgent need to develop another type of antibiotics.
Focusing on LecA
She works collaboratively with two international groups to do the virtual screening to sort the compounds to be used with LecA crystal

Bruker talk: immuno peptidomic analysis of SarCoV2 infected lung epithelial cells

Using mass spec to understand the immunology in many diseases, also the basic knowledges
HLA-class II -- TCR on CD4+ Tcell
HLA-class I -- TCR on CD8+ Tcell
HLAs have diverse peptide binding preferences
Challenging of MS

Low abundance peptides which we might miss -- we can tackle by using lots of biopsy or highly sensitive mass-spec

COVID19 vaccine

Genetic engineered spike area protein to improve the efficiency of SAR-CoV vaccine, namely hexapro
It has actually been used at clinical trial phase 1 already.
To produce hybridoma from mice, he collaborates with SI group to produce the neutralizing antibody from his genetically engineered plasmid.
mRNA is very labile once it is injected into the body
He modified mRNA to enhance the stability.

18-Nov-2021

Plant-based COVID19 vaccine

It is great that she has another colleague who has a economic/policy background, thus her team is very successful in establishing the startup company.
Just learning that there are tobacco strains which genetically modify to have a glycosylation pattern similar to humans.
I ask this question: How do you maintain the plant strain? Do you have a QC system to check from time to time? Do we have to buy seeds from abroad? We characterize the plant and have to qc using DNA barcode technique to make sure all the plants we use are Nicotiana benthamiana. We don’t have to buy the seed. We can produce the seed in our facility. -- Thus, this approach seems to be very sustainable.

LC-MS support COVID-19 fight

In terms of vaccines, LC-MS can help with vaccine development, especially to check whether the mRNA is integrity or whether there are any contaminations.
To analyze the glycosylation at the viral spike.

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